PROJECT SUMMARY Aortic valve stenosis (AVS) is a progressive disease characterized by excessive deposition of extracellular matrix (ECM) proteins at the aortic valve, leading to increased valve stiffness and eventual heart failure. Unfortunately, clinicians depend on valve replacement surgery for therapy, where surgery may lead to complications including stroke and major bleeding around the replacement. This proposal seeks to develop a non-surgical alternative for treating AVS using functionalized hydrogel nanoparticles, or nanogels, capable of targeting the fibrotic microenvironment of the aortic valve and delivering the mechanical cues necessary to repair the stiffened tissue. In the proposed research, nanogels will be engineered with varying modulus and functionality to target the diseased aortic valve in vivo. Nanogels will be designed to target the microenvironment surrounding valvular interstitial cells (VICs), which are fibroblast-like cells located at valve leaflets and serve to regulate ECM deposition and remodeling in response to valve injury. Upon injury, VICs are known to transition to myofibroblasts, which lead to the aberrant accumulation of ECM proteins and promote increased aortic valve stiffness. The delivery of nano-scale stiffness cues to the diseased aortic valve is hypothesized to halt or reverse activation of valvular interstitial cell (VIC) to myofibroblasts, serving as a strategy to modulate AVS progression. In Aim I, poly(ethylene glycol) (PEG) nanogels of varying moduli will be fabricated using thiol-ene crosslinking chemistry and suspension polymerization techniques. Thiol-ene chemistry allows for the facile coupling of fibrotic valve targeting peptides (V-CAM and fibrin binding) and fluorophores on the nanogel, offering a tunable platform to alter nanogel functionality for in vitro and in vivo applications. PEG nanogels of varying modulus will be characterized using atomic force microscopy (AFM), dynamic light scattering, and transmission electron microscopy. Aim II will evaluate VIC-nanogel interactions that lead to the reversal of the myofibroblast phenotype in vitro. VICs will be cultured on bulk PEG substrates of varying stiffness to obtain quiescent VICs and activated myofibroblast populations. Then, VICs will be treated with nanogels to evaluate activation/reversal of the myofibroblast phenotype as a function of nanogel modulus, using markers of VIC activation including intracellular alpha-smooth muscle actin (?-SMA) expression. Furthermore, the role of nanogels in mediating PI3K/AKT and TGF-? fibrotic signaling pathway expression will be characterized using whole transcriptome sequencing. Aim III will assess nanogel targeting in vivo using a hypercholesterolemic/hypertensive (HC/HT) murine model of spontaneously occurring fibrotic AVS. Nanogels will be delivered intravenously to assess targeting to the diseased aortic valve as a function of nanogel dose concentration and targeting peptide. Valve histology and staining for ?-SMA, AFM, and survival studies using HC/HT mice will determine the effects of nanogel modulus in mediating valve fibrosis. The proposed research will elucidate a new role for nano-scale mechanical cues to mediate VIC phenotype, as well as provide a critical link toward a non-surgical therapy for AVS.